This invention relates to ligand-targeted emulsions that incorporate biologically active agents on or in their particle surface, and more particularly, to such novel emulsions that are especially useful for the treatment of disease with bioactive agents that have improved risk/benefit profiles when applied specifically to selected cells, tissues or organs.
As used herein, the following terms have the definitions set forth:
Direct conjugation of ligand to the emulsion particle refers to the preparation of a ligand-particle complex before administration wherein the ligand is either adsorbed through ionic, electrostatis, hydrophobic or other noncovalent means to the particle surface (e.g. acylated-antibody), or chemically linked to the surface through covalent bonds to a component of the lipid surface such as a xe2x80x9cprimer materialxe2x80x9d (e.g. thio-ether or ester bond), or intrinsically incorporated into the lipid surfactant membrane as a component of the membrane (e.g. a lipid derivatized to a peptiodomimetic agent).
Indirect conjugation refers to the use of avidin biotin where the complex is formed in vivo in two or more steps. An example would be giving the biotinylated antibody first, followed by avidin, and followed by the biotinylated emulsion particle. Any other sequential multistep chemical linking system that could be utilized in vivo is envisioned to produce the same end result, i.e. the close and specific apposition of the emulsion particle to a targeted cell or tissue surface.
Primer material refers to any constituent or derivatized constituent incorporated into the emulsion lipid surfactant layer that could be chemically utilized to form a covalent bond between the particle and a targeting ligand or a component of the targeting ligand (if it has subunits).
Prolonged association of the emulsion particle with the surface of the targeted cell or tissue is in contradistinction to the transient interaction that an unbound particle, existing free in extracellular body fluids, would achieve. By binding the particle to the cell surface, the continued circulation of the nanoparticle through the body is halted. The affixed particle is able to interact with the target cell surface over an extended period of time. The exact amount of time may be variable, but is meant to exceed that of more transient nontargeted contact between particles and cell surfaces by orders of magnitude.
Surfactant is a term derived from SURFace ACTive AgeNT. A Surfactant is a compound that contains a hydrophilic and a hydrophobic segment. When added to water or solvents, a surfactant reduces the surface tension of the systems for the following purposes emulsifying or dispersing in the present application. Our preferred surfactants are phospholipids and cholesterol but include those lipids that are mentioned in our previous application and the additional detergents specified in our invention disclosure.
Ligand is a molecule that binds to another molecule, used in this application to refer to a small targeting molecule that binds specifically to another molecule on a biological surface separate and distinct from the emulsion particle itself. The reaction does not require nor exclude a molecule that donates or accepts a pair of electrons to form a coordinate covalent bond with a metal atom of a coordination complex.
Emulsion technology is very old and distinct from the more modern liposome technology. This is exemplified by the prolific research and patent literature involving liposomes since the 1963 report by Bangham (Physical structure and behavior of lipids and lipid enzymes., Adv Lipid Res, 1963; 1:65-104). Bangham originally characterized emulsions as xe2x80x9ceither temporary or permanent dispersions of oils or hydrophobic material in water or vice versaxe2x80x9d and liposomes as xe2x80x9c . . . xe2x80x98myelinsxe2x80x99 and xe2x80x98myelinicsxe2x80x99 . . . irrevocably associated with the structures obtained when certain phospholipids are dispersed in water. . . . The unit structure is a biomolecular tube of lipids, separated from its adjacent concentric tube by a layer of water.xe2x80x9d In later years liposomes have been elegantly described as xe2x80x9cvesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecules . . . (which) form spontaneously when these lipids are dispersed in aqueous media. . . . The liposome membrane forms a bilayer structure which is in principle identical to the lipid portion of natural cell membranes.xe2x80x9d Liposomes may be prepared by a variety of techniques and have single or multiple membrane layers. They are distinctly different and more complex than emulsions.
Drugs can be incorporated into liposomes within either the internal aqueous phase or within one or more of the lipid bilayer membranes and liposomes can be coupled to ligands of various types. Because of the bilayer nature of a liposome membrane, lipophilic drugs incorporate into both the inner and outer leaflets of the bilayer. Drugs, bound to the inner leaflet layer are unavailable for immediate delivery by contact facilitated delivery as opposed to lipid encapsulated emulsions. For multilamellar liposomes, most of the drug will be internalized within the liposome and not readily available for contact facilitated delivery to a target cell. To extend circulatory half-life, liposomes have been modified with polymerized lipids or the addition of polyethylene glycol to enhance in vivo survivability. Both modifications protect the particles from lipid exchange with other cells and lipoproteins.
xe2x80x9cAn emulsion is a heterogeneous system, consisting of at least one immiscible liquid intimately dispersed in another in the form of droplets, whose diameters, in general, exceed 0.1xcexc. Such systems possess a minimal stability, which may be accentuated by such additives as surface-active agents, finely-divided solids, etc.xe2x80x9d (Becher P. Emulsion: Theory and Practice, New York, N.Y.; Reinhold Publishing Corporation; 1965) xe2x80x9cThe phase which is present in the form of finely divided droplets is called the dispersion or internal phase; the phase which forms the matrix in which these droplets are suspended is called the continuous or external phase . . . Surface active or other agents which are added to increase stability . . . are known as emulsifiers or emulsifying agents. Stability is also increased by mechanical devices such as simple stirrers, homogenizers or colloid mills.xe2x80x9d
Liquid perfluorocarbon emulsions are specialized formulations with various medical and oxygen transport applications. They are especially useful medically as contrast media, for various biological imaging modalities such as nuclear magnetic resonance, ultrasound, x-ray, computed tomography, F-magnetic resonance imaging, and position emission tomography, as oxygen transport agents or xe2x80x9cartificial bloods,xe2x80x9d in the treatment of heart attack, stroke, and other vascular obstructions, as adjuvants to coronary angioplasty and in cancer radiation treatment and chemotherapy. The fluorocarbon emulsion can be used to deliver drugs and medicines soluble in or transportable by the emulsion.
Long et al. U.S. Pat. No. 4,987,154 discloses that fluorocarbon emulsions can deliver therapeutic agents, medicines and drugs throughout the body, tissue and organs by at least two modes: 1) within the fluorocarbon phase or 2) by complexing of the agent, medicine or drug with the surfactant membrane. Long et al. cite examples of medicines, drugs and therapeutic agents that can be dissolved in the fluorocarbon including diazepam, cyclosporin, rifampin, clindamycin, isoflurane, halothane and enflurane. Examples of medicines, therapeutic agents and drugs that do not dissolve in fluorocarbon, but can be complexed with, for example, a lecithin membrane include mannitol, tocopherol, streptokinase, dexamethasone, prostaglandin E, interleukin, gentamycin and cefoxitin. Antibiotics may be delivered transcutaneously through the skin when added to a fluorocarbon emulsion. Furthermore, proteins such as thrombolytic agents, hormones or enzymes can be transported and delivered by fluorocarbon emulsions.
Delivery of drugs as described by Long et al. and others depend upon the encapsulated drug being more slowly metabolized and eliminated from the circulation than free drug. In other cases, the encapsulated particles are sequestered into organs and cells involved with the normal metabolism and clearance of particles and foreign matter from the body, a process referred to as passive targeted delivery. The opportunity to conjugate ligands to perfluorocarbon emulsions for the purpose of contact facilitated delivery of bioactive agents was not envisioned by Long et al.
We have previously reported a novel ligand-targeted, lipid-encapsulated nongaseous perfluorocarbon emulsion useful for ultrasound, magnetic resonance and nuclear imaging applications (U.S. Pat. Nos. 5,690,907, 5,780,010, 5,958,371 and 5,989,520). The perfluorocarbon emulsion is produced through microfluidization techniques, and is robustly stable to handling, pressure, atmospheric exposure, heat and shear. In the early phases of development, we coupled a pretargeted biotinylated ligand to a biotinylated version of the emulsion nanoparticle through avidin-biotin interactions. Subsequently, we adopted a direct ligand conjugation approach using monoclonal F(ab) fragments to facilitate future clinical implementations.
The emulsion nanoparticles have long circulatory half-lives due to their small size and inherent in vivo stability without further modification of their outer lipid surfaces with polyethylene glycol or incorporation of polymerized lipids. Surfactant modifications often detract from targeting efficacy in order to extend circulatory persistence. The in vivo clearance of the nanoparticles was measured in dogs by quantification of the blood perfluorocarbon content with an estimated half-life of one hour. Preliminary data suggest that this novel agent will persist bound to tissue for hours, and dependent upon location, even days.
Millbrath et al [U.S. Pat. No. 5,401,634] disclose fluorochemical emulsions comprised of a fluorochemical discontinuous phase and aqueous continuous phase with at least one specific binding species immobilized on the droplets. The emulsions can include a xe2x80x9cprimer materialxe2x80x9d to couple specific binding species to the fluorochemical droplets. The emulsions may be used in diagnostic procedures or biochemical reactors where binding of the immobilized specific binding species to its binding partner is desired. The droplets were envisioned to incorporate a species (e.g. dye) that is detectable by spectrophotometric, fluorometric or colormetric means. These inventors were focused upon in vitro applications and were unconcerned with in vivo targeted drug delivery. Moreover, they never conceived of the benefits of contact facilitated drug or gene delivery achieved through ligand-targeted emulsion technology.
Magdassi et al, (published PCT application WO 95/03829) described the production and use of ligand-targeted oil emulsions in which drug is xe2x80x9cdissolved, dispersed or solubilized inside the oil droplet, creating a novel drug targeting systemxe2x80x9d. The targeted particles provide an oil encapsulated depot of drug at the target site. Subsequent breakdown of the particles releases drug to the interstium. The agent is diluted in extracellular fluids and can migrate from the interstitium to the target cell. Magdassi et al. do not conceive of the utility of contact facilitated delivery of drug or genes from an outer surfactant layer to target tissues. They do not recognize the importance or advantages of a biocompatible phospholipid surfactant layer amenable to exchanging constituents with the target cell membrane. Contact facilitated drug delivery with ligand-targeted emulsions places all of the drug into a monolayer surrounding the particle, ready to interact with the target surface. Mobility of the phospholipid monolayer over the particle surface allows drug from all regions of the layer to migrate and interact with target cell surfaces. Encapsulating drug within the particle, as described by Magdassi et al. isolates the agent from the particle surface and prevents contact facilitated delivery to the target cell membrane.
Unger et al (U.S. Pat. No. 5,542,935) have described therapeutic delivery systems for site-specific delivery of bioactive agents using gas-filled perfluorocarbon microspheres. The microspheres contain a temperature activated gaseous precursor that becomes a gas upon activation at a selected temperature. Once the microspheres have been introduced into the patient""s body, a therapeutic compound may be targeted to specific tissues through the use of sonic energy, microwave energy, magnetic energy, or hyperthermia, which is directed to the target area and causes the microspheres to rupture and release the therapeutic compound. Perfluorocarbons preferred include perfluoromethane, perfluoroethane, perfluorobutane, perfluoropentane, perfluorohexane; even more preferably perfluoroethane, perfluoropentane, perfluoropropane, and perfluorobutane. Unger et al. notes that the localization of these particles for cavitation can be improved with conjugated ligand and the process of active targeting.
There remains a need for improved bioactive agent delivery systems which provide enhanced efficiency of such bioactive agents to targeted tissues, cells or organs.
Among the several objects of the invention may be noted the provision of novel compositions and methods for use in delivering bioactive agents to targeted tissues or cells; the provision of such compositions which provide enhanced delivery of bioactive agents to targeted tissues or cells; the provision of such methods which provides enhanced intermingling and exchange of lipid components from one lipid surface to the other thereby facilitating the exchange of bioactive agents within or on the bioactive agent/emulsion surface to the target cells or tissues; and the provision of such compositions and methods which may be readily practiced. Other objects will be in part apparent and in part pointed out hereinafter.
Briefly, in one aspect, the present invention is directed to a composition for use in delivering a bioactive agent to targeted tissues or cells comprising:
(a) site-specific targeting ligand;
(b) a lipid encapsulated oil in water emulsion; and
(c) a bioactive agent in or on the surface of the outer lipid monolayer of said emulsion, said ligand being conjugated directly or indirectly to said emulsion and the composition providing facilitated delivery of the bioactive agent through prolonged association and increased contact of the ligand-bound, lipid encapsulated emulsion particles with the lipid bilayer of said target tissues or cells. The invention is also directed to a method for improved delivery of a bioactive agent to targeted tissues or cells comprising administering the above-noted compositions to said tissues or cells.